Biomedical subjects

R A Morgan

Publications and source records attributed to R A Morgan.

At least 19 recordsLinked to original sources

PubMed2002

Inhibition of HIV-1 replication by novel lentiviral vectors expressing transdominant Rev and HIV-1 env antisense.

Retroviral vectors expressing transdominant negative mutants of Rev (TdRev) inhibit HIV-1 replication by preventing the nuclear export of unspliced viral transcripts, thus inhibiting the synthesis of Gag-Pol, Env and reducing the levels of genomic RNA available for packaging. Due to these effective mechanisms of inhibition, production of HIV-1-based lentiviral vectors expressing TdRev has been difficult. Here we describe HIV-based vectors in which expression of TdRev is negatively regulated by Rev expression. In these vectors, we maintained the wild-type HIV-1 Tat/Rev exons and intron configuration and its mode of splicing regulation. The second Rev exon was mutated to encode TdRev. Inhibition of TdRev expression by Rev during vector production yields high titer vector preparations. A second vector containing an additional anti-HIV gene (env-antisense) was constructed by flipping a 1.2-kb env fragment contained within the Tat/TdRev intron. SupT1 cells and primary CD4+ lymphocytes transduced with these vectors inhibit HIV-1 replication and show a preferential advantage for survival. Although these vectors are poorly mobilized to secondary target cells by wild-type HIV-1, they reduce the infectivity of the wild-type virions escaping inhibition.

CD4-Positive T-Lymphocytes↗

PubMed2001

Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems.

Short interfering RNAs (siRNAs) are double-stranded RNAs of approximately 21-25 nucleotides that have been shown to function as key intermediaries in triggering sequence-specific RNA degradation during posttranscriptional gene silencing in plants and RNA interference in invertebrates. siRNAs have a characteristic structure, with 5'-phosphate/3'-hydroxyl ends and a 2-base 3' overhang on each strand of the duplex. In this study, we present data that synthetic siRNAs can induce gene-specific inhibition of expression in Caenorhabditis elegans and in cell lines from humans and mice. In each case, the interference by siRNAs was superior to the inhibition of gene expression mediated by single-stranded antisense oligonucleotides. The siRNAs seem to avoid the well documented nonspecific effects triggered by longer double-stranded RNAs in mammalian cells. These observations may open a path toward the use of siRNAs as a reverse genetic and therapeutic tool in mammalian cells.

Animals↗

PubMed2001

Malignant biliary disease: percutaneous interventions.

Interventional radiologists have an important role in the management of patients with malignant biliary obstruction. This article describes the techniques for percutaneous biliary drainage, insertion of biliary endoprostheses, and the management of occluded biliary endoprostheses. Most procedures are performed by using fluoroscopic guidance alone. Ultrasound is also a useful modality for guiding biliary drainage, particularly drainage of the left biliary ducts. Patients should be treated by internal drainage if possible. Metallic endoprostheses can be inserted at the time of the initial biliary drainage procedure. Plastic tubes should be inserted a few days after biliary drainage because of their relatively large size compared with metallic stents. Occluded plastic stents should be replaced. Blocked metallic stents should be treated either by placement of additional overlapping metallic stents or by placement of plastic stents within the metallic stent lumen.

Bile Duct Neoplasms↗

PubMed2001

Development of an avian leukosis-sarcoma virus subgroup A pseudotyped lentiviral vector.

We are using avian leukosis-sarcoma virus (ALSV) vectors to generate mouse tumor models in transgenic mice expressing TVA, the receptor for subgroup A ALSV. Like other classical retroviruses, ALSV requires cell division to establish a provirus after infection of host cells. In contrast, lentiviral vectors are capable of integrating their viral DNA into the genomes of nondividing cells. With the intention of initiating tumorigenesis in resting, TVA-positive cells, we have developed a system for the preparation of a human immunodeficiency virus type 1 (HIV-1)-based lentiviral vector, pseudotyped with the envelope protein of ALSV subgroup A (EnvA). The HIV(ALSV-A) vector retains the requirement for TVA on the surface of target cells and can be produced at titers of 5 x 10(3) infectious units (IU)/ml. By inserting the central polypurine tract (cPPT) from the HIV-1 pol gene and removing the cytoplasmic tail of EnvA, the pseudotype can be produced at titers approaching 10(5) IU/ml and can be concentrated by ultracentrifugation to titers of 10(7) IU/ml. HIV(ALSV-A) also infects embryonic fibroblasts derived from transgenic mice in which TVA expression is driven by the beta-actin promoter. In addition, this lentivirus pseudotype efficiently infects these fibroblasts after cell cycle arrest, when they are resistant to infection by ALSV vectors. This system may be useful for introducing genes into somatic cells in adult TVA transgenic animals and allows evaluation of the effects of altered gene expression in differentiated cell types in vivo.

Alpharetrovirus↗

PubMed2001

Inhibition of human immunodeficiency virus type 1 (HIV-1) replication by HIV-1-based lentivirus vectors expressing transdominant Rev.

Retrovirus vectors expressing transdominant-negative mutants of Rev (TdRev) inhibit human immunodeficiency virus type 1 (HIV-1) replication by preventing the nuclear export of unspliced viral transcripts, thus inhibiting the synthesis of Gag-Pol, Env, and genomic RNA. The use of HIV-1-based vectors to express TdRev would have the advantage of allowing access to nondividing hematopoietic cells. It would also provide additional levels of protection by sequestering the viral regulatory proteins Tat and Rev, competing for encapsidation into wild-type virions, and inhibiting reverse transcription. Here we describe HIV-1-based vectors that express TdRev. These vectors contain mutations in the splicing signals or replacement of the Rev-responsive element by the simian retrovirus type 1 constitutive transport element, making them less sensitive to the inhibitory effects of TdRev. In addition, overexpression of Rev and the use of an HIV-1 helper plasmid that drives high levels of Gag-Pol synthesis were used to transiently overcome the inhibition by TdRev of the synthesis of Gag-Pol during vector production. SupT1 cells transduced with these vectors were more resistant to HIV-1 replication than cells transduced with Moloney murine leukemia virus-based vectors expressing TdRev. Furthermore, we show that these vectors can be mobilized by the wild-type virus, reducing the infectivity of virions escaping inhibition and conferring protection against HIV-1 replication to previously untransduced cells.

Blotting, Southern↗

PubMed2001

Percutaneous treatment of pseudoaneurysms using fibrin adhesive.

This study was performed to assess the safety and efficacy of treatment of iatrogenic pseudoaneurysms with fibrin adhesive. 28 patients with iatrogenic pseudoaneurysms were treated. The first 20 patients had either at least one previously unsuccessful treatment of ultrasound guided compression repair (UGCR), a contraindication to UGCR or could not tolerate the procedure. Diagnosis was confirmed on ultrasound and angiography and a balloon inflated across the neck of the pseudoaneurysm. Fibrin adhesive was injected under ultrasound control directly into the pseudoaneurysm sac. The balloon was deflated after 15 min and check ultrasound and angiography performed, with a further ultrasound at 24 h. The subsequent eight patients underwent embolisation as the first line treatment without use of a protective balloon. 16 of the first 20 patients (80%) and all of the subsequent 8 patients had successful embolisation of the pseudoaneurysm after a single treatment. Two pseudoaneurysms failed to thrombose despite two treatments in one case and three treatments in the other, and both required surgery. The only significant complication was the development of local cellulitis in one patient. In conclusion, embolisation of iatrogenic aneurysms with fibrin adhesive is a safe and effective treatment. It should be considered as an alternative to surgery and UGCR.

Adult↗

PubMed2000

Potent inhibition of human immunodeficiency virus type 1 replication by conditionally replicating human immunodeficiency virus-based lentiviral vectors expressing envelope antisense mRNA.

We describe an HIV-based lentiviral vector that expresses a 1-kb antisense mRNA directed against the HIV-1 mRNAs containing env sequences. The expression of antisense env mRNAs (envAS) does not inhibit the synthesis of p24 expressed from the HIV-1 helper plasmid used to package the vector, as this helper has a deletion in the env gene. This allows the production of high-titer VSV-G pseudotyped lentiviral particles. In challenge experiments using unselected populations of SupT1 cells transduced with this vector, a complete inhibition of HIV-1 replication was observed for long periods of in vitro culture, even at high HIV-1 infectious doses. The potent inhibition of HIV-1 replication by this vector correlated with a low occurrence of mobilization of the vector to previously untransduced cells. The infectivity of the wild-type HIV-1 that escapes inhibition was highly inhibited, suggesting that the vector is providing HIV-1 inhibition of replication not only due to its antisense effect but also by competing for encapsidation and mobilization to noninfected cells.

Blotting, Northern↗

PubMed2000

Efficient gene transfer to human peripheral blood monocyte-derived dendritic cells using human immunodeficiency virus type 1-based lentiviral vectors.

Dendritic cells (DCs) are potent antigen-presenting cells and are capable of activating naive T cells. Gene transfer of tumor antigen and cytokine genes into DCs could be an important strategy for immunotherapeutic applications. Dendritic cells derived from peripheral blood monocytes do not divide and are therefore poor candidates for gene transfer by Moloney murine leukemia virus (Mo-MuLV)-based retroviral vectors. Lentiviral vectors are emerging as a powerful tool for gene delivery into dividing and nondividing cells. A three-plasmid expression system pseudotyped with the envelope from vesicular stomatitis virus (VSV-G) was used to generate lentiviral vector particles expressing enhanced green fluorescent protein (EGFP). Peripheral blood monocyte-derived DCs were cultured in the presence of GM-CSF and IL-4 and transduced with lentiviral or Mo-MuLV-based vectors expressing EGFP. FACS analysis of lentiviral vector-transduced DCs derived either from normal healthy volunteers or from melanoma patients demonstrated transduction efficiency ranging from 70 to 90% compared with 2-8% using Mo-MuLV-based vectors pseudotyped with VSV-G. Comparison of lentiviral vectors expressing EGFP driven by CMV or human PGK promoters showed similar levels of transgene expression. Lentiviral vector preparations produced in the absence of HIV accessory proteins transduced DCs at efficiencies equal to vectors produced with accessory proteins. Alu-HIV-1 LTR PCR demonstrated the genomic integration of the lentiviral vector in the transduced DCs. Transduced cells showed characteristic dendritic cell phenotype and strong allostimulatory capacity and maintained the ability to respond to activation signals such as CD40 ligand and lipopolysaccharide. These results provide evidence that lentiviral vectors are efficient tools for gene transfer and expression in monocyte-derived DCs that could be useful for immunotherapeutic applications.

Alu Elements↗

PubMed2000

Lentiviral-mediated gene transfer into human lymphocytes: role of HIV-1 accessory proteins.

Resting lymphocytes are refractory to gene transfer using Moloney murine leukemia virus (MMLV)-based retroviral vectors because of their quiescent status. Recently, it has been shown that lentiviral vectors are capable of transferring genes into nondividing and terminally differentiated cells. We used human immunodeficiency virus type-1 (HIV-1)-based vectors expressing enhanced green fluorescent protein (EGFP) driven by different promoters (CMV, MPSV, or PGK) and investigated their ability to transduce human T- and B-cell lines, as well as resting or activated primary peripheral and umbilical cord blood lymphocytes. The effects of the presence or the absence of HIV-1 accessory proteins (Vif, Vpr, Vpu, and Nef) in the vector system were also assessed. Flow cytometry analysis showed no differences in the ability of these vectors of transferring the reporter gene into lymphocytic lines and mitogen-stimulated primary lymphocytes in the presence or the absence of HIV-1 accessory proteins (APs). Similarly, viral supernatants generated in the presence of accessory genes could efficiently transduce various subsets of resting lymphocytes and provide long-term expression of the transgene. No significant transduction-induced changes in cell activation or cycling status were observed and Alu-HIV-1 long terminal repeat polymerase chain reaction (LTR PCR) analysis demonstrated integration of the vector sequences at the molecular level. In contrast, in the absence of HIV-1 APs, lentiviral vectors failed to integrate and express the transgene in resting lymphocytes. These results show that transduction of primary resting lymphocytes with HIV-1-based vectors requires the presence of viral accessory proteins. (Blood. 2000;96:1309-1316)

B-Lymphocytes↗

PubMed2000

dsRNA-mediated gene silencing in cultured Drosophila cells: a tissue culture model for the analysis of RNA interference.

RNA interference (RNAi) is a form of post-transcriptional gene silencing that has been described in a number of plant, nematode, protozoan, and invertebrate species. RNAi is characterized by a number of features: induction by double stranded RNA (dsRNA), a high degree of specificity, remarkable potency and spread across cell boundaries, and a sustained down-regulation of the target gene. Previous studies of RNAi have examined this effect in whole organisms or in extracts thereof; we have now examined the induction of RNAi in tissue culture. A screen of mammalian cells from three different species showed no evidence for the specific down-regulation of gene expression by dsRNA. By contrast, RNAi was observed in Drosophila Schneider 2 (S2) cells. Green fluorescent protein (GFP) expression in S2 cells was inhibited in a dose-dependent manner by transfection of dsRNA corresponding to gfp when GFP was expressed either transiently or stably. This effect was structure- and sequence-specific in that: (1) little or no effect was seen when antisense (or sense) RNA was transfected; (2) an unrelated dsRNA did not reduce GFP expression; and (3) dsRNA corresponding to gfp had no effect on the expression of an unrelated target transgene. This invertebrate tissue culture model should allow facile assays for loss of function in a well-defined cellular system and facilitate further understanding of the mechanism of RNAi and the genes involved in this process.

Animals↗

PubMed2000

Modified human immunodeficiency virus-based lentiviral vectors display decreased sensitivity to trans-dominant Rev.

As a first step toward the development of HIV-based conditionally replicating defective interfering particles expressing trans-dominant Rev (TdRev), we studied whether mutation of the splicing signals and replacement of the RRE by the SRV-1 CTE would render these vectors less sensitive to TdRev. Vectors with mutations in the splicing signals (SD-/RRE+) yielded high titers (5 X 10(6) CFU/ml) and showed higher levels of cytoplasmic unspliced mRNA than the corresponding SD+/RRE+ vectors either in the absence of Rev, in the presence of TdRev, or in the presence of both TdRev and Rev. Proviral copies of SD-/RRE+ vectors were rescued more efficiently than SD+/RRE+ vectors when TdRev was expressed. Vectors with the SRV-1 CTE (SD+/CTE+ and SD-/CTE+) expressed high levels of cytoplasmic unspliced mRNA in the absence of Rev expression. Titers obtained with the SD-/CTE+ vectors (10(6) CFU/ml) were higher than the titers obtained with SD+/CTE+ vectors. We also tested the effect of other structural modifications such as the orientation of the expression cassette and the presence of the central polypurine tract (cPPT/CTS). We show that an expression cassette cloned in the reverse orientation with respect to the LTRs or elimination of the cPPT/CTS element severely affected vector titers. We also demonstrated that these vectors can be efficiently mobilized from their proviral state by HIV trans-complementing functions, and transduced into secondary target cells without suffering any genomic rearrangement.

Base Sequence↗

PubMed2000

Evaluation of recombinant alphaviruses as vectors in gene therapy.

Alphavirus vectors based on Sindbis virus and Semliki Forest virus (SFV) were characterized as potential gene transfer vectors. Initial studies were performed using vectors engineered to transfer either lacZ or green fluorescent protein (GFP). High levels of gene transfer were achieved in human primary fibroblasts, BHK and 293T cells, with low levels of transduction observed in more than 20 other target cells. Alphavirus-based expression was generally very high, but transient in every cell type. Replication-competent alphavirus was never detected in SFV preparations but could be produced by Sindbis-based vectors at a frequency of up to 3 x 10(-3) infectious units per ml. We constructed a human clotting factor IX (hFIX) cDNA-containing Sindbis virus and compared it with hFIX cDNA-harboring adenoviral and retroviral vectors. In most cases, hFIX levels obtained with Sindbis vector were initially at least an order of magnitude higher than those obtained with other viral vectors. These data demonstrate that alphavirus vectors compare favorably with adenovirus vectors as systems to promote high-level transient gene expression and should be considered as an alternative vector for gene transfer and potential gene therapy studies.

Alphavirus↗

PubMed2000

Improved titers of HIV-based lentiviral vectors using the SRV-1 constitutive transport element.

The development of lentiviral vectors that use Rev-independent mechanisms of nuclear export for their genomic RNA could facilitate the construction of novel anti-HIV vectors. We have improved the titers of Rev-independent lentiviral vectors having the SRV-1 CTE by mutating the major splice donor and acceptor sites present in the vector and by relocalization of the CTE sequences adjacent to the HIV-1 3'LTR. These two modifications have additive beneficial effects on vector titers and packaging efficiency. Packaging these CTE+ vectors expressing marker genes with a Rev-dependent HIV-1 helper vector yields higher titers than are obtained using a Rev-dependent lentiviral vector.

Acquired Immunodeficiency Syndrome↗

PubMed1999

Adenovirus-mediated expression of human coagulation factor IX in the rhesus macaque is associated with dose-limiting toxicity.

We used a first-generation adenovirus vector (AVC3FIX5) to assess whether human factor IX could be expressed and detected in the rhesus macaque, which we have shown does not make high-titer antibodies to human factor IX protein. Three animals received 1 x 10(10) to 1 x 10(11) plaque-forming units per kilogram by intravenous injection. Human factor IX was present within 24 hours of vector administration and peaked 4 days later at 4,000 ng/mL in the high-dose recipient, and lower levels were seen in the intermediate-dose recipient. No human factor IX was detected in the low-dose recipient's plasma. Serum cytokine analysis and early hypoferremia suggested a dose-dependent acute-phase response to the vector. Human factor IX was detectable in rhesus plasma for 2 to 3 weeks for the high- and intermediate-dose recipients, but disappeared concomitant with high-titer antihuman factor IX antibody development. There was substantial, dose-dependent, dose-limiting liver toxicity that was manifest as elevated serum transaminase levels, hyperbilirubinemia, hypoalbuminemia, and prolongation of clotting times. Of particular interest was prolongation of the thrombin clotting time, an indicator of decreased fibrinogen or fibrinogen dysfunction. All evidence of liver toxicity resolved except for persistent hypofibrinogenemia in the high-dose recipient, indicating possible permanent liver damage. Our data suggest a narrow therapeutic window for first-generation adenovirus-mediated gene transfer. The development of antihuman factor IX antibodies and abnormalities of fibrinogen in the rhesus macaque is of concern for application of adenovirus (or other viral) vectors to hemophilia gene therapy.

Adenoviridae↗

PubMed1999

Gene therapy: lessons learnt from the past decade. Interview by Clare Thompson.

Explore the source record for details and available documents.

Acquired Immunodeficiency Syndrome↗

PubMed1999

Production of minigene-containing retroviral vectors using an alphavirus/retrovirus hybrid vector system.

In an attempt to increase the synthesis of human clotting factors VIII and IX in transduced cells, optimized expression cassettes containing genomic genelike elements (minigenes) were assembled. Plasmid DNA containing factor VIII or factor IX minigenes and driven by three human cellular promoters (albumin, factor IX, PGK) or the strong viral promoter RSV-LTR were electroporated into TE671 and HepG2 cell lines, and clotting factor levels were determined by ELISA. In comparison with a parallel transfection of MLV-LTR-promoted retroviral vector plasmid DNAs, the PGK- and RSV-LTR-promoted minigene constructs produced equal or greater amounts of clotting factor proteins. A factor IX minigene cassette was cloned into the retrovirus-based gene transfer vector LN (in both forward and reverse orientations) and the minigene vector was introduced into the Phoenix retroviral packaging cell line. Analysis of neo(r) cells demonstrated that insertion of a factor IX minigene into the retroviral vector LN resulted in rearrangement of the factor IX sequence and loss of factor IX expression in the Phoenix packaging cell line. The same factor IX minigene was then inserted into an alphavirus/retrovirus hybrid vector that facilitates the synthesis of retroviral vector RNA in the cytoplasm of cells. Alphavirus/retrovirus virions were produced and used to transduce the Phoenix retroviral vector packaging cell line. The cytoplasmically produced factor IX minigene-containing retroviral vectors were collected and used to transduce TE671 cells. Analysis of transduced cells demonstrated stable transfer of the minigene and expression of factor IX.

Alphavirus↗

PubMed1999

The rhesus macaque as an animal model for hemophilia B gene therapy.

We have determined the 2905 nucleotide sequence of the rhesus macaque factor IX complementary DNA (cDNA) and found it to be greater than 95% identical to that of the human factor IX cDNA. The cDNA has a large 3' untranslated region like the human cDNA, but unlike the human cDNA has two polyadenylation sites 224 nucleotides apart that are used for transcription of the messenger RNA. The deduced amino acid sequence is greater than 97% identical to that of human factor IX, differing in only 11 of 461 amino acids in the complete precursor protein. We found a single silent polymorphism in the nucleotide sequence at the third position of the codon for asparagine at position 167 in the secreted protein (AAC/AAT). All residues subject to posttranslational modifications in the human protein are also found in the rhesus factor IX sequence. The high degree of homology between the rhesus and human factor IX proteins suggested the possibility that the human factor IX protein might be nonimmunogenic in the rhesus. We tested the immunogenicity of human factor IX in three rhesus macaques by repeated intravenous injections of monoclonal antibody-purified, plasma-derived human factor IX over the course of more than a year and assessed the recovery and half-life of the infused protein, as well as in vitro indicators of antihuman factor IX antibodies. Human factor IX recovery and half-life remained unchanged over the course of a year in the three animals studied, and aPTT mixing studies showed no evidence for neutralizing antihuman factor IX antibodies. An outbred, nonhuman primate model that permits assessment of the level and duration of factor IX expression as well as vector safety would complement the use of other (mouse and canine) hemophilia B animal models in current use for the development of gene therapy for hemophilia B.

Amino Acid Sequence↗

PubMed1999

Inhibition of human immunodeficiency virus type 1 by Tat/Rev-regulated expression of cytosine deaminase, interferon alpha2, or diphtheria toxin compared with inhibition by transdominant Rev.

A retroviral vector was designed to express toxic proteins only in the presence of the HIV-1 Rev and/or Tat protein(s). The design of this vector incorporates an HIV-specific expression cassette that consists of three elements: the U3R region of the HIV-1 IIIB LTR provides the promoter and Tat-responsive element, a modified intron derived from the human c-src gene facilitates the splicing of inserted genes, and the HIV-1 RRE region enhances the transport of unspliced mRNAs. To further limit potential readthrough transcription, the expression cassette was inserted in the reverse transcriptional orientation relative to the retroviral vector LTR. Three different genes, interferon alpha2, diphtheria toxin (DT-A), and cytosine deaminase, were inserted into this vector. Tat and Rev inducibility was demonstrated directly by a >300-fold induction of interferon production and functionally by a decrease in colony-forming units when a Tat and Rev expression vector was titered on HeLa cells harboring the inducible DT-A cassette. The Tat-inducible cytosine deaminase gene was tested in the Sup-T1 T cell line and shown to inhibit HIV-1 production only when engineered cells were grown in the presence of 5-fluorocytosine. To test the ability of this system to inhibit HIV-1 infection in bulk PBL cultures, a series of transduction and challenge experiments was initiated with both the interferon and DT-A vectors. Protection against infection was documented against three HIV strains in PBLs. Last, the interferon and DT-A vectors were compared with a vector encoding a transdominant Rev protein and were shown to mediate equal or greater inhibition of HIV-1.

Blotting, Northern↗